1. Field of the Invention
The present invention relates to a method of analyzing a laminated structure, which includes cell populations forming a layered structure, in a state of a three-dimensional structure by imaging (image) and a method of evaluating a laminated structure for use in corneal transplantation using the image analysis method.
Priority is claimed on Japanese Patent Application No. 2012-195938, filed Sep. 6, 2012, the content of which is incorporated herein by reference.
2. Description of the Related Art
All patents, patent applications, patent publications, scientific articles, and the like, which will hereinafter be cited or identified in the present application, will hereby be incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.
In recent years, for the purpose of regenerating damaged biological tissues or organs, regenerative medicine using stem cells has attracted attention. A treatment method in which a structure obtained by culturing stem cells from a patient and forming a three-dimensional structure similar to a tissue is transplanted is excellent in that, unlike organ transplantation, there are no rejection and donor insufficiency. Actually, treatment in which a laminated structure formed by culturing epithelial stem cells, such as corneal epithelial stem cells and oral mucosal stem cells from a patient, is transplanted to a cornea so as to regenerate the cornea is put into practical use.
In general, stem cells are transplanted as a laminated structure in which a layer having differentiated cells is formed above a layer of stem cells, or a laminated structure in which a plurality of single-layered structures having only a layer of stem cells formed are superimposed. Each cell in the laminated structure transplanted to the biological tissue is proliferated and differentiated after engraftment, and as a result, the biological tissue is regenerated. Whether or not a damaged biological tissue is sufficiently generated by transplantation of a laminated structure for transplantation depends on the quality of the laminated structure for transplantation. For this reason, the evaluation of the quality of the laminated structure for transplantation is important.
In regard to the evaluation of the quality of the laminated structure for corneal transplantation, for example, in “Tissue Engineering: Part C, Hayashi and seven others, 2009, Vol. 16, No. 4, pp. 553 to 560” (hereinafter, referred to as Non-Patent Literature 1), first, the laminated structure for corneal transplantation is divided into halves. Then, one half is divided into individual cells, and the total number of cells, the number of living cells, and the number of cytokeratin-positive cells (that is, the number of epithelial cells) are quantitatively measured by flow cytometry. Frozen sections (having a thickness is about 10 um) are created from the other half, each section is subjected to HE staining or immunostaining for p63, AE5, ZO-1, and MUC16, the states of the cells are examined, and the results are synthesized to evaluate the quality.
In the evaluation method described in Non-Patent Literature 1, since the frozen sections are analyzed, only a considerably small part of the laminated structure for corneal transplantation can be evaluated, and multi-dimensional analysis is impossible. That is, it is not possible to analyze the positional relationship of the cells in the laminated structure for corneal transplantation. It also takes a lot of time and effort to divide the laminated structure for corneal transplantation. In addition, a worker should have proficiency in producing frozen sections, and in many cases, there is a difference between samples.
The invention provides a method of more simply analyzing images of a laminated structure, which includes cell populations forming a layered structure, while maintaining information regarding the positional relationship between cells in the laminated structure, and a method of evaluating a laminated structure for corneal transplantation using the image analysis method.